Liposome-based homogeneous luminescence resonance energy transfer.
There is an increasing need for developing simple assay formats for biomedical screening purposes. Assays on cell membranes have become important in studies of receptor-ligand interactions and signal pathways. Here luminescence energy transfer was studied on liposomes containing europium ion chelated to 4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione and trioctylphosphine oxide. Energy transfer efficiency was characterized with biotin-streptavidin interaction, and a model assay concept for a homogeneous time-resolved luminescence resonance energy transfer (LRET) assay was developed. Acceptor-labeled streptavidin was bound to biotinylated lipids on the liposomes, leading to close proximity of the LRET pair. The liposome-based LRET assay was optimized for dye incorporation and concentration, biotinylation degree, liposome size, and kinetics. Sensitivity for a competitive biotin assay was at a picomolar range with a coefficient of variation from 7 to 20%. The developed lipid membrane-based system was feasible in separation free LRET assay concept with high sensitivity, indicating that the assay principle can potentially be used for biologically more relevant target molecules.
Pihlasalo
Sari
S
Laboratory of Biophysics, University of Turku, FIN-20520 Turku, Finland. sari.pihlasalo@ut.u.fi
Hara
Mika
M
Hänninen
Pekka
P
Slotte
J Peter
JP
Peltonen
Jouko
J
Härmä
Harri
H
D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D004735:Energy Transfer; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism; D005063:Europium / Q000737:chemistry / Q000378:metabolism; D007659:Ketones / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D009281:Naphthalenes / Q000737:chemistry / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism
C534544:4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione; D004852:Epoxy Compounds; D007659:Ketones; D008081:Liposomes; D009281:Naphthalenes; D009943:Organophosphorus Compounds; D001360:Avidin; D005063:Europium; C007366:diepoxybutane; D001710:Biotin; C044965:trioctyl phosphine oxide